Our previous studies of protein synthesis in the squid giant axon system revealed that de novo protein synthesis occurs in neuronal cell bodies but not from axoplasm from the giant axon. Recent studies have shown that the squid giant axon contains elements of the protein synthetic machinery, i.e., polysomes and mRNAs in addition to tRNA. These investigators suggested that protein synthesis can occur in the axonal compartment of the giant axon system. We have generated specific antibodies to squid neurofilament (NF) proteins to test this hypothesis. Since we have shown NF mRNA is present in axons, we used these antibodies in biosynthesis/immunoprecipitation experiments using the squid giant axon to test the hypothesis of axonal protein synthesis. These experiments confirmed robust biosynthesis of NF proteins in squid stellate ganglia, but we failed to detect any NF protein biosynthesis in the giant axon. We also examined the localization of NFs in adult tissues and during neural development using NF protein-specific antibodies. These studies showed that NFs were present in adult neural tissues, primarily in selected fibers, with giant axons showing the most robust expression. After the first neurons differentiated at stage 22, immunoblots showed NF60 and NF70 immunoreactive proteins at all stages. The NF 220 subunit, however, was not detected in immunoblots at any developmental stage. Phosphorylated NF 220 immunoreactivity, however, though absent in immunoblots was first seen in selected fibers of the stellate ganglia at stage 25, increasing thereafter, in all giant fibers until hatching (stage 30). The stellate ganglion is the first neural tissue to acquire a mature neurofilament complement (i.e., phosphorylated NF 220), shortly before the onset of jet-propelled escape behavior. The temporal pattern of expression of the NFS during development resembled that seen in vertebrates, i.e., the smaller NFs appeared before the larger subunits in most neural tissues. In the squid, the expression pattern seems to depend upon a post transcriptional regulation of a single gene rather than by transcriptional regulation of three independent genes as in vertebrates.